JP7740802B2 - Storage container for cell-containing liquid and storage liquid - Google Patents

Description

The present invention relates to a storage container for storing a cell-containing liquid. The present invention also relates to a storage liquid used for storing a cell-containing liquid.

Body fluids such as blood, plasma, serum, and urine contain cell-free DNA (cfDNA). It is known that cfDNA is present at higher concentrations in the body fluids of people with malignant tumors and infectious diseases compared to healthy individuals (Non-Patent Document 1).

In recent years, tests using cfDNA as a sample have been conducted in areas such as cancer and genetic diseases. Tests using cfDNA as a sample place less of a burden on patients than tests that require the collection of diseased tissue from the patient.

Additionally, maternal blood contains free DNA (cell-free fetal DNA, cffDNA) derived from the fetus (Non-Patent Document 2). Prenatal genetic testing uses cffDNA as a sample.

Tests using cfDNA as a sample are difficult to perform unless performed at specialized institutions equipped with specialized equipment such as qPCR devices or Next Generation Sequencers (NGS). For this reason, cell-containing fluids such as blood collected at hospitals and other facilities must be transported to specialized institutions, and a certain number of days are required from collection to analysis. During this time, the cell-containing fluid is stored in a designated container.

The following Patent Documents 1 and 2 describe methods for stably recovering cfDNA by stabilizing cells, particularly white blood cells, present in whole blood.

Furthermore, Patent Document 3 listed below discloses a serum-free vitrification cryopreservation solution for animal cells that contains at least one water-soluble cellulose-based cryoprotectant selected from the group consisting of hydroxypropyl cellulose, hydroxypropyl methylcellulose, and hydroxyethyl methylcellulose. Patent Document 3 also describes that the vitrification cryopreservation solution preferably contains a specific cell membrane-permeable cryoprotectant or a specific cell membrane-impermeable cryoprotectant.

WO2013/123030A1 CN107083382A JP 2011-111406 A

Karen-Lise Garm Spindler, Ane L.Appelt, Niels Pallisgaard, et al. Cell-free DNA in individuals healthy, noncancerous disease and strong prognostic value in colorectal cancer. Int. J. Cancer: 135, 2984-2991 (2014) Alberry M, Maddocks D, Jones M, et al. Free fetal DNA in maternal plasma in anembryonic pregnancies: confirmation that the origin is the trophoblast. Prenat Diagn. 2007 May; 27(5): 415-8

Tests are being conducted using cfDNA contained in cell-containing fluids such as blood as a sample. The cell-containing fluid, such as blood, is collected in a storage container containing a preservative solution and then transported to a specialized institution equipped with a qPCR device or next-generation sequencer. While transportation to the specialized institution may be carried out in a frozen environment, it is preferable to transport the fluid at room temperature (e.g., 15°C to 25°C) in order to reduce transportation costs and to prevent any problems during transportation.

However, in storage containers containing conventional preservation solutions, the storage stability of the cfDNA contained in the cell-containing solution is likely to decrease when the cell-containing solution is stored at room temperature. For example, in conventional storage containers, cfDNA may be degraded by DNases contained in the cell-containing solution during storage at room temperature. Furthermore, in conventional storage containers, cells contained in the cell-containing solution may be destroyed or killed during storage at room temperature, causing the genomic DNA (gDNA) in the cells to leak into the cell-containing solution. In conventional storage containers containing EDTA, EDTA can suppress cfDNA degradation to some extent by inhibiting DNase activation, but it cannot suppress cell death, etc.

The method described in Patent Document 1 can improve the storage stability of cfDNA contained in cell-containing fluid to a certain extent. However, the inventors have found that even with the method described in Patent Document 1, the formaldehyde released by the formaldehyde donor compound crosslinks nucleic acids together or nucleic acids and proteins, thereby increasing the apparent fragment length of cfDNA and reducing the storage stability of cfDNA. Furthermore, in prenatal genetic testing, it is necessary to distinguish between fetal-derived cfDNA (cffDNA) and maternal-derived cfDNA. CffDNA fragments are degraded during passage through maternal tissues such as the placenta, resulting in shorter fragment lengths than maternal-derived cfDNA. Prenatal genetic testing distinguishes between cffDNA and cfDNA based on the difference in fragment length. Therefore, if formaldehyde crosslinks cffDNA and cfDNA, causing changes in the fragment lengths of cffDNA and cfDNA, this can have a significant impact on test results.

Furthermore, even with the method described in Patent Document 2 above, it is difficult to sufficiently increase the storage stability of cfDNA.

Furthermore, when a conventional vitrification cryopreservation solution such as that described in Patent Document 3 is used to preserve a cell-containing fluid, such as a body fluid, the vitrification cryopreservation solution is excessively diluted by the cell-containing fluid, preventing the vitrification cryopreservation solution from fully exerting its effects. This makes it difficult to improve the preservation stability of the cfDNA contained in the cell-containing fluid.

As such, using conventional methods, it is difficult to sufficiently increase the storage stability of cfDNA when the cell-containing liquid is stored at room temperature.

An object of the present invention is to provide a storage container for cell-containing liquid that can improve the storage stability of cfDNA contained in the cell-containing liquid even when stored at room temperature. Another object of the present invention is to provide a storage liquid that can improve the storage stability of cfDNA contained in the cell-containing liquid even when stored at room temperature.

According to a broad aspect of the present invention, there is provided a cell-containing fluid storage container for storing a predetermined amount of cell-containing fluid, the container comprising: a container body; and a preservation fluid contained within the container body. The preservation fluid comprises a cell membrane-permeable compound having a molecular weight of 100 or less that does not freeze at 0°C. When the predetermined amount of cell-containing fluid is collected in the cell-containing fluid storage container and mixed with the preservation fluid to obtain a mixed fluid X, the content of the cell membrane-permeable compound in the mixed fluid X is 1 vol% or more and 5 vol% or less. Alternatively, the preservation fluid comprises a cell membrane-impermeable compound having a molecular weight of 300 or more. When the predetermined amount of cell-containing fluid is collected in the cell-containing fluid storage container and mixed with the preservation fluid to obtain a mixed fluid X, the content of the cell membrane-impermeable compound in the mixed fluid X is 0.5 μmol/L or more and 5 μmol/L or less.

In a particular aspect of the cell-containing liquid storage container of the present invention, the cell-containing liquid storage container has the first configuration, and the cell membrane-permeable compound is a polyhydric alcohol.

In a specific aspect of the cell-containing liquid storage container of the present invention, the cell-containing liquid storage container has the first configuration, and the cell membrane-permeable compound is ethylene glycol, propylene glycol, glycerin, dimethyl sulfoxide, acetamide, 1,3-propanediol, or butylene glycol.

In a particular aspect of the cell-containing liquid storage container of the present invention, the cell-containing liquid storage container has the second configuration, and the cell membrane-impermeable compound is a compound having a molecular weight of 1,000 or more.

In a specific aspect of the cell-containing liquid storage container of the present invention, the cell-containing liquid storage container has the second configuration, and the cell membrane-impermeable compound is polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, a polysaccharide, a polysaccharide derivative, a sugar alcohol, or ficoll.

In a particular aspect of the cell-containing fluid storage container of the present invention, the cell-containing fluid storage container has the first configuration, and the storage fluid contains a formaldehyde donor compound as a compound different from both the cell membrane-permeable compound and the cell membrane-impermeable compound.

In a particular aspect of the cell-containing fluid storage container of the present invention, the storage fluid contains a formaldehyde donor compound as a compound different from both the cell membrane-permeable compound and the cell membrane-impermeable compound, and when solution Y' is obtained by mixing 7.4 g of sodium hypochlorite with 1 L of physiological saline, and an amount of solution Y' equal to a predetermined amount of cell-containing fluid to be stored in the cell-containing fluid storage container is collected in a cell-containing fluid storage container, and mixed solution Y' with the storage fluid is obtained, the formaldehyde content in mixed solution Y is 100 mg/L or more and 400 mg/L or less.

In a particular aspect of the cell-containing liquid storage container of the present invention, the formaldehyde donor compound is DMDM hydantoin or 1-hydroxymethyl-5,5-dimethylhydantoin.

In a particular aspect of the cell-containing fluid storage container of the present invention, when a volume of saline equal to a predetermined volume of cell-containing fluid to be stored in the cell-containing fluid storage container is collected in the cell-containing fluid storage container, and the saline and the storage fluid are mixed to obtain a mixed fluid Z, the osmotic pressure of the mixed fluid Z is 300 mOsm/L or more and 1100 mOsm/L or less.

In a particular aspect of the storage container for cell-containing fluid of the present invention, the storage fluid contains an osmotic pressure adjuster as a compound different from both the cell membrane-permeable compound and the cell membrane-impermeable compound, and the osmotic pressure adjuster is glucose or sodium chloride.

In a particular aspect of the storage container for cell-containing liquid of the present invention, the storage liquid contains a compound with chelating activity as a compound different from both the cell membrane-permeable compound and the cell membrane-impermeable compound, the compound with chelating activity contains EDTA, and the content of EDTA in the mixed liquid X is 4 mmol/L or more and 7 mmol/L or less.

In a particular aspect of the storage container for cell-containing liquid of the present invention, the storage liquid contains a buffer as a compound different from both the cell membrane-permeable compound and the cell membrane-impermeable compound, the buffer contains glycine, and the content of glycine in the mixed liquid X is 0.5 w/v% or less.

In a particular aspect of the storage container for cell-containing liquid of the present invention, the storage container for cell-containing liquid has the first configuration and the second configuration.

In a particular aspect of the storage container for cell-containing liquid of the present invention, the cell-containing liquid is blood.

According to a broad aspect of the present invention, there is provided a preservation solution used for preserving a cell-containing fluid, the preservation solution having a third configuration in which the preservation solution contains a cell membrane-permeable compound having a molecular weight of 100 or less, which does not freeze at 0°C, and the content of the cell membrane-permeable compound is 2 vol% or more and 50 vol% or less, or a fourth configuration in which the preservation solution contains a cell membrane-impermeable compound having a molecular weight of 300 or more, and the content of the cell membrane-impermeable compound is 1.0 μmol/L or more and 100 μmol/L or less.

In a particular aspect of the preservation solution of the present invention, the preservation solution has the third configuration, and the cell membrane-permeable compound is a polyhydric alcohol.

In a specific aspect of the preservation solution of the present invention, the preservation solution has the third configuration, and the cell membrane-permeable compound is ethylene glycol, propylene glycol, glycerin, dimethyl sulfoxide, acetamide, 1,3-propanediol, or butylene glycol.

In a particular aspect of the preservation solution of the present invention, the preservation solution has the fourth configuration, and the cell membrane-impermeable compound is a compound having a molecular weight of 1,000 or more.

In a specific aspect of the preservation solution of the present invention, the preservation solution has the fourth configuration, and the cell membrane-impermeable compound is polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, a polysaccharide, a polysaccharide derivative, a sugar alcohol, or ficoll.

In a particular aspect of the preservation solution of the present invention, the preservation solution includes a formaldehyde donor compound as a compound different from both the cell membrane-permeable compound and the cell membrane-impermeable compound.

In a particular aspect of the preservative solution of the present invention, the formaldehyde donor compound is DMDM hydantoin or 1-hydroxymethyl-5,5-dimethylhydantoin.

In a specific aspect of the preservation solution of the present invention, the preservation solution contains an osmotic agent as a compound different from both the cell membrane-permeable compound and the cell membrane-impermeable compound, and the osmotic agent is glucose or sodium chloride.

In a particular aspect of the preservation solution of the present invention, the preservation solution contains a compound having chelating activity as a compound different from both the cell membrane-permeable compound and the cell membrane-impermeable compound, and the compound having chelating activity includes EDTA.

In a particular aspect of the preservation solution of the present invention, the preservation solution contains a buffer as a compound different from both the cell membrane-permeable compound and the cell membrane-impermeable compound, and the buffer contains glycine.

In a particular aspect of the preservation solution of the present invention, the preservation solution has the third configuration and the fourth configuration.

In a particular aspect of the preservation solution of the present invention, the cell-containing solution is blood.

The cell-containing fluid storage container of the present invention is a container for storing a predetermined amount of cell-containing fluid, and comprises a container body and a storage fluid contained within the container body. The cell-containing fluid storage container of the present invention has the following first or second configuration. First configuration: The storage fluid contains a cell membrane-permeable compound with a molecular weight of 100 or less that does not freeze at 0°C, and when the predetermined amount of cell-containing fluid is collected in the cell-containing fluid storage container and mixed with the storage fluid to obtain a mixed fluid X, the content of the cell membrane-permeable compound in the mixed fluid X is 1 vol% or more and 5 vol% or less. Second configuration: The storage fluid contains a cell membrane-impermeable compound with a molecular weight of 300 or more, and when the predetermined amount of cell-containing fluid is collected in the cell-containing fluid storage container and mixed with the storage fluid to obtain a mixed fluid X, the content of the cell membrane-impermeable compound in the mixed fluid X is 0.5 μmol/L or more and 5 μmol/L or less. The storage container for cell-containing liquid according to the present invention has the above-described configuration, and therefore can improve the storage stability of cfDNA contained in the cell-containing liquid even when stored in a room temperature environment.

The preservation solution of the present invention is a preservation solution used to preserve a cell-containing solution. The preservation solution of the present invention has the following third or fourth configuration. Third configuration: Contains a cell membrane-permeable compound that has a molecular weight of 100 or less and does not freeze at 0°C, and the content of the cell membrane-permeable compound is 2 vol% or more and 50 vol% or less. Fourth configuration: Contains a cell membrane-impermeable compound that has a molecular weight of 300 or more, and the content of the cell membrane-impermeable compound is 1.0 μmol/L or more and 100 μmol/L or less. Because the preservation solution of the present invention has the above configurations, it can enhance the storage stability of cfDNA contained in the cell-containing solution even when stored at room temperature.

1(a) to (c) show the results of image analysis of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Example 1. 2(a) and (b) show the results of image analysis of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Example 2. 3(a) to (c) show the results of image analysis of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Comparative Example 1, and FIG. 3(d) shows the results of image analysis of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Comparative Example 4. 4(a) and (b) show the results of image analysis of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Comparative Example 2. 5(a) to (c) show the image analysis results of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Comparative Example 3. 6(a) to (c) show the results of image analysis of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Examples 3 to 5. 7(a) to (c) show the results of image analysis of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Examples 6 to 8. 8(a) to (d) show the results of image analysis of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Examples 9 to 12. 9(a) and (b) show the results of image analysis of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Example 13. 10(a) and (b) show the results of image analysis of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Example 14. 11(a) and (b) show the results of image analysis of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Example 15.

The present invention is described in detail below.

The cell-containing fluid storage container (hereinafter sometimes abbreviated as "storage container") of the present invention is a container for storing a predetermined amount of cell-containing fluid, and comprises a container body and a storage fluid contained within the container body. The cell-containing fluid storage container of the present invention has the following first or second configuration.

First configuration: The preservation liquid contains a cell membrane-permeable compound having a molecular weight of 100 or less and that does not freeze at 0°C, and when the predetermined amount of cell-containing liquid is collected in a cell-containing liquid storage container and mixed with the preservation liquid to obtain a mixed liquid X, the content of the cell membrane-permeable compound in the mixed liquid X is 1 vol% or more and 5 vol% or less.

Second configuration: The preservation solution contains a cell membrane impermeable compound having a molecular weight of 300 or more, and when the predetermined amount of cell-containing solution is collected in a cell-containing solution storage container and mixed with the preservation solution to obtain a mixed solution X, the content of the cell membrane impermeable compound in the mixed solution X is 0.5 μmol/L or more and 5 μmol/L or less.

The preservation solution of the present invention is a preservation solution used to preserve a cell-containing solution. The preservation solution of the present invention has the following third or fourth configuration.

Third configuration: Contains a cell membrane-permeable compound having a molecular weight of 100 or less and that does not freeze at 0°C, and the content of the cell membrane-permeable compound is 2 vol% or more and 50 vol% or less.

Fourth configuration: Contains a cell membrane impermeable compound having a molecular weight of 300 or more, and the content of the cell membrane impermeable compound is 1.0 μmol/L or more and 100 μmol/L or less.

In the storage container and storage solution of the present invention, the cell-containing solution is stored in a mixed state with the preservation solution. Because the storage container and storage solution of the present invention have the above-described configuration, the storage stability of the cfDNA contained in the cell-containing solution can be enhanced even when stored at room temperature. The storage container and storage solution of the present invention can enhance the storage stability of the cfDNA contained in the cell-containing solution, even when stored, for example, in an environment between 15°C and 25°C. The present invention can effectively prevent cells contained in the cell-containing solution from being destroyed or killed during storage at room temperature, thereby preventing the gDNA contained in the cells from being mixed into the cell-containing solution. Therefore, the present invention can enhance the storage stability of the cfDNA. For example, the present invention can stably store cfDNA contained in the cell-containing solution at 25°C for one day or more (e.g., seven days).

The cell-containing fluid is a fluid that contains cells. Examples of the cell-containing fluid include body fluids, such as blood, urine, and cerebrospinal fluid. Blood contains cells such as white blood cells.

It is preferable that the cell-containing liquid is blood, as this will more effectively demonstrate the effects of the present invention.

The storage container of the present invention may have the first configuration, the second configuration, or both the first and second configurations. The storage container of the present invention may have only one of the first and second configurations, or both. From the perspective of more effectively achieving the effects of the present invention, it is preferable that the storage container of the present invention have both the first and second configurations.

The preservation solution of the present invention may have the third configuration described above, the fourth configuration described above, or both the third and fourth configurations. The preservation solution of the present invention may have only one of the third and fourth configurations described above, or may have both. From the perspective of more effectively achieving the effects of the present invention, it is preferable that the preservation solution of the present invention have both the third and fourth configurations described above.

(preservation solution)
The above preservative solution is liquid at 25°C.

The preservation solution contains a cell membrane-permeable compound with a molecular weight of 100 or less that does not freeze at 0°C, or a cell membrane-impermeable compound with a molecular weight of 300 or more. The preservation solution may contain the cell membrane-permeable compound, the cell membrane-impermeable compound, or both the cell membrane-permeable compound and the cell membrane-impermeable compound.

Whether a compound is a cell membrane permeable compound or a cell membrane impermeable compound is usually determined by the compound's molecular weight, charge, polarity, etc. Uncharged compounds are usually classified as cell membrane permeable compounds because they can permeate cell membranes by passive diffusion. Furthermore, for example, charged compounds are usually classified as cell membrane impermeable compounds because they cannot permeate cell membranes by passive diffusion.

<Cell membrane permeable compound>
The preservation solution preferably contains a cell membrane-permeable compound having a molecular weight of 100 or less and not freezing at 0°C. The cell membrane-permeable compound is a compound that can permeate cell membranes. When the storage container has the first configuration, the preservation solution contains the cell membrane-permeable compound. When the storage solution has the third configuration, the preservation solution contains the cell membrane-permeable compound. By using the cell membrane-permeable compound, cells can be efficiently dehydrated and the cell membrane-permeable compound can be efficiently retained within the cells. Therefore, it is possible to effectively prevent cells from being destroyed or dying during storage, which could lead to the gDNA in the cells being mixed into the cell-containing solution. The cell membrane-permeable compound is also known as a cryoprotectant for cells. The cell membrane-permeable compound may be used alone or in combination of two or more.

Examples of the above-mentioned cell membrane-permeable compounds include ethylene glycol, propylene glycol, glycerin, dimethyl sulfoxide, acetamide, 1,3-propanediol, and butylene glycol.

The cell membrane-permeable compound is preferably ethylene glycol, propylene glycol, glycerin, dimethyl sulfoxide, acetamide, 1,3-propanediol, or butylene glycol, and more preferably ethylene glycol, propylene glycol, glycerin, or dimethyl sulfoxide. Furthermore, the cell membrane-permeable compound is preferably a polyhydric alcohol. In this case, the effects of the present invention can be even more effectively achieved.

The content of the cell membrane-permeable compound in the preservation solution is not particularly limited, as long as the content of the cell membrane-permeable compound in the mixed solution X is 1 vol% or more and 5 vol% or less. The content of the cell membrane-permeable compound in the preservation solution can be changed as appropriate depending on the mixing ratio of the cell-containing solution and the preservation solution, etc.

The content of the cell membrane-permeable compound in the preservation solution is preferably 2 vol% or more, more preferably 10 vol% or more, even more preferably 20 vol% or more, preferably 50 vol% or less, more preferably 40 vol% or less, and even more preferably 30 vol% or less. When the content of the cell membrane-permeable compound is above the above-mentioned lower limit and below the above-mentioned upper limit, it becomes easier to adjust the content of the cell membrane-permeable compound in the mixture of the cell-containing solution and the preservation solution (mixed solution X) to fall within the above range, and as a result, the effects of the present invention can be more effectively exerted.

<Cell membrane impermeable compound>
The preservation solution preferably contains a cell membrane impermeable compound having a molecular weight of 300 or more. When the preservation container has the second configuration, the preservation solution contains the cell membrane impermeable compound. When the preservation solution has the fourth configuration, the preservation solution contains the cell membrane impermeable compound. By using the cell membrane impermeable compound, cell aggregation can be effectively suppressed and the protective effect of the cell membrane can be enhanced. Therefore, it is possible to effectively prevent cells from being destroyed or killed during storage, which would result in the gDNA in the cells being mixed into the cell-containing solution. The cell membrane impermeable compound may be used alone or in combination of two or more types.

Examples of the cell membrane-impermeable compounds include polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, polysaccharides, polysaccharide derivatives, sugar alcohols, and ficoll. Examples of the polysaccharides include cellulose, sucrose, and dextran. Examples of the polysaccharide derivatives include hydroxypropyl cellulose.

From the viewpoint of more effectively achieving the effects of the present invention, the cell membrane impermeable compound is preferably polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, polysaccharides, polysaccharide derivatives, sugar alcohol, or ficoll. From the viewpoint of even more effectively achieving the effects of the present invention, the cell membrane impermeable compound is more preferably polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, dextran, or hydroxypropyl cellulose, and even more preferably polyvinylpyrrolidone, polyethylene glycol, or polyvinyl alcohol.

From the viewpoint of achieving the effects of the present invention, the molecular weight of the cell membrane impermeable compound is 300 or more. When the structural formula of the cell membrane impermeable compound can be identified, the molecular weight of the cell membrane impermeable compound refers to the molecular weight that can be calculated from the structural formula; when the structural formula cannot be identified, the molecular weight refers to the weight average molecular weight.

The molecular weight (weight average molecular weight) of the cell membrane impermeable compound is preferably 1,000 or more, more preferably 10,000 or more, even more preferably 100,000 or more, and preferably 4,000,000 or less, more preferably 1,000,000 or less. When the molecular weight (weight average molecular weight) of the cell membrane impermeable compound is above the above lower limit and below the above upper limit, the effects of the present invention can be exerted even more effectively.

The above weight average molecular weight means the weight average molecular weight in polystyrene equivalent terms measured by gel permeation chromatography (GPC).

The content of the cell membrane impermeable compound in the preservation solution is not particularly limited, as long as the content of the cell membrane impermeable compound in the mixed solution X is 0.5 μmol/L or more and 5 μmol/L or less. The content of the cell membrane impermeable compound in the preservation solution can be changed as appropriate depending on the mixing ratio of the cell-containing solution and the preservation solution, etc.

The content of the cell membrane impermeable compound in the preservation solution is preferably 1.0 μmol/L or more, more preferably 5 μmol/L or more, and preferably 100 μmol/L or less, more preferably 50 μmol/L or less. When the content of the cell membrane impermeable compound is above the above lower limit and below the above upper limit, it becomes easier to adjust the content of the cell membrane impermeable compound in the mixture of the cell-containing solution and the preservation solution (mixed solution X) to fall within the above range, and as a result, the effects of the present invention can be more effectively exerted.

<Formaldehyde donor compound>
The preservation solution preferably contains a formaldehyde donor compound as a compound different from both the cell membrane-permeable compound and the cell membrane-impermeable compound. The formaldehyde donor compound is a compound capable of liberating formaldehyde. By using the formaldehyde donor compound, when the cell-containing solution and the preservation solution are mixed, the action of formaldehyde liberated from the formaldehyde donor compound crosslinks cellular membrane proteins, thereby enhancing cell stability. Conventional preservation solutions containing a formaldehyde donor compound are prone to changes in the fragment length of cfDNA contained in the cell-containing solution during storage at room temperature. In contrast, the present invention makes it possible to reduce changes in the fragment length of cfDNA even when the preservation solution contains a formaldehyde donor compound. The formaldehyde donor compound may be used alone, or two or more types may be used in combination.

Examples of the formaldehyde donor compounds include hydantoin compounds, imidazolidinyl urea, diazolidinyl urea, hexamethylenetetramine, N,N''-methylenebis-[N'-(3-hydroxymethyl-2,5-diaoxo-4-imidazolidinyl)urea], tertiary amine compounds, secondary amine compounds, and primary amine compounds.

Examples of the above hydantoin compounds include DMDM hydantoin, 1-hydroxymethyl-5,5-dimethylhydantoin, 1,3-dimethylol-5,5-hydantoin, and 1,3-dichloro-5,5-dimethylhydantoin.

From the viewpoint of more effectively achieving the effects of the present invention, the above-mentioned formaldehyde donor compound is preferably a hydantoin compound, and more preferably DMDM hydantoin or 1-hydroxymethyl-5,5-dimethylhydantoin.

The content of the formaldehyde donor compound in the preservation solution is not particularly limited. The content of the formaldehyde donor compound in the preservation solution can be changed as appropriate, for example, so that the formaldehyde content in the mixed solution Y described below satisfies the range described below. The content of the formaldehyde donor compound in the preservation solution can be changed as appropriate depending on the mixing ratio of the cell-containing solution to the preservation solution, the type of formaldehyde donor compound, etc.

<Osmolality adjuster>
The preservation solution preferably contains an osmotic pressure adjuster as a compound different from both the cell membrane-permeable compound and the cell membrane-impermeable compound. By using the osmotic pressure adjuster, the osmotic pressure of the mixed solution can be effectively increased. The osmotic pressure adjuster may be used alone or in combination of two or more.

Examples of the above-mentioned osmotic pressure adjusting agents include inorganic ions such as sodium chloride and potassium chloride, and sugars such as glucose and sucrose.

From the viewpoint of more effectively exerting the effects of the present invention, the osmotic pressure adjuster is preferably glucose, sucrose, or sodium chloride, and more preferably glucose or sodium chloride.

The content of the osmotic pressure adjuster in the preservative solution is not particularly limited. The content of the osmotic pressure adjuster in the preservative solution can be changed as appropriate, for example, so that the osmotic pressure of the mixed solution Z described below falls within the range described below.

<Buffering Agent>
The preservation solution preferably contains a buffer as a compound different from both the cell membrane-permeable compound and the cell membrane-impermeable compound. The buffer may be used alone or in combination of two or more.

Examples of the buffering agent include phosphates such as glycine, sodium citrate, sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate; carbonates such as sodium carbonate and sodium bicarbonate; borates such as sodium borate; carboxylic acids, dicarboxylic acids, carboxylic acid derivatives, hydroxycarboxylic acids, aniline, aniline derivatives, amino acids, amine compounds, imidazole compounds, alcohol compounds, ethylenediaminetetraacetic acid, pyrophosphoric acid, pyridine, cacodylic acid, glycerol phosphate, 2,4,6-collidine, N-ethylmorpholine, morpholine, 4-aminopyridine, ammonia, ephedrine, hydroxyproline, piperidine, and tris(hydroxymethyl)aminomethane.

From the viewpoint of effectively exerting a buffering effect, it is preferable that the buffer contains glycine, and it is preferable that it is glycine.

The content of the buffering agent in the preservation solution is not particularly limited as long as it is a content that can exert a buffering effect. The content of the buffering agent in the preservation solution can be changed as appropriate depending on the mixing ratio of the cell-containing solution and the preservation solution, etc.

<Compounds with chelating activity>
The preservation solution preferably contains a compound having a chelating activity as a compound different from both the cell membrane-permeable compound and the cell membrane-impermeable compound. Because DNase is activated by magnesium ions, the use of a compound having a chelating activity can effectively suppress the degradation of cfDNA in the cell-containing solution. When the cell-containing solution is blood, the preservation solution preferably contains an anticoagulant as the compound having a chelating activity. In this case, in addition to effectively suppressing the degradation of cfDNA, it can also effectively suppress blood coagulation during storage. The compound having a chelating activity may be used alone, or two or more types may be used in combination.

Examples of the above anticoagulants include ethylenediaminetetraacetic acid (EDTA), citric acid, and glycol ether diaminetetraacetic acid (EGTA).

From the standpoint of more effectively suppressing the degradation of cfDNA and, if the cell-containing liquid is blood, more effectively suppressing blood coagulation during storage, it is preferable that the anticoagulant contains EDTA, and is preferably EDTA.

The content of the compound having chelating activity in the preservation solution can be changed as appropriate depending on the mixing ratio of the cell-containing solution to the preservation solution, the type of compound having chelating activity, etc.

<Other ingredients>
The preservation solution may contain components other than the cell membrane-permeable compound, the cell membrane-impermeable compound, the formaldehyde donor compound, the osmotic pressure adjuster, the buffer, and the compound having a chelating activity, such as water and a pH adjuster.

The preservative solution preferably contains water. The water content of the preservative solution (100% by weight) is preferably 30% by weight or more, more preferably 40% by weight or more, and preferably 60% by weight or less, more preferably 50% by weight or less.

The osmotic pressure of the preservation solution is preferably 300 mOsm/L or more, more preferably 600 mOsm/L or more, and preferably 4000 mOsm/L or less, more preferably 3000 mOsm/L or less. When the osmotic pressure of the preservation solution is above the above lower limit and below the above upper limit, the effects of the present invention can be more effectively exerted.

The osmotic pressure of the above preservative solution can be measured using an osmometer (e.g., Advanced Instruments' Osmometer 3250).

The pH of the preservative solution is preferably 6.0 or higher, more preferably 7.0 or higher, and preferably 8.0 or lower, more preferably 7.5 or lower. When the pH is above the lower limit and below the upper limit, the effects of the present invention can be more effectively achieved.

(Storage container for cell-containing solution)
The preservation container according to the present invention is a container for preserving a predetermined amount of cell-containing fluid. When the preservation container according to the present invention has the first configuration, when the predetermined amount of cell-containing fluid is collected in the preservation container and the cell-containing fluid and the preservation fluid are mixed to obtain a mixed fluid X, the content of the cell membrane-permeable compound in the mixed fluid X is within a specific range.

When the storage container of the present invention has the second configuration, when the predetermined amount of cell-containing liquid is collected in the storage container and the cell-containing liquid and the storage liquid are mixed to obtain a mixed liquid X, the content of the cell membrane-impermeable compound in the mixed liquid X is within a specific range.

In the storage container of the present invention, the predetermined amount of cell-containing liquid is mixed with the storage liquid contained in the storage container to obtain the mixed liquid X.

Specifically, the above-mentioned mixed solution X is prepared as follows.

A predetermined amount of cell-containing fluid is collected in the storage container. For example, if the storage container is designed to collect 5 mL of blood, 5 mL of blood is collected in the storage container. The collected predetermined amount of cell-containing fluid and the storage fluid are mixed by inversion to obtain mixed fluid X.

In a storage container having the first configuration, the content of the cell membrane-permeable compound in the mixed solution X is 1 vol% or more and 5 vol% or less. That is, in a storage container having the first configuration, the content of the cell membrane-permeable compound in 100% by volume of the mixed solution is 1 vol% or more and 5 vol% or less. In a storage container having the first configuration, the content of the cell membrane-permeable compound in the mixed solution X is within the above range, so that water within the cells can be effectively dehydrated and the cell membrane-permeable compound can be effectively retained within the cells. Therefore, it is possible to effectively prevent cells from being destroyed or killed during storage, which could result in gDNA in the cells being mixed into the cell-containing solution.

In the mixed solution X, the content of the cell membrane-permeable compound is preferably 2 vol% or more, and preferably 4 vol% or less. When the content of the cell membrane-permeable compound is above the above lower limit and below the above upper limit, the cells can be more effectively dehydrated and the cell membrane-permeable compound can be more effectively retained within the cells. This more effectively prevents the cells from being destroyed or dying during storage, which could lead to the gDNA in the cells being mixed into the cell-containing solution.

In a storage container having the second configuration, the content of the cell membrane impermeable compound in the mixed solution X is 0.5 μmol/L or more and 5 μmol/L or less. In a storage container having the second configuration, the content of the cell membrane impermeable compound in the mixed solution X is within the above range, which effectively suppresses cell aggregation and enhances the protective effect of the cell membrane. Therefore, it is possible to effectively prevent cells from being destroyed or killed during storage, which could result in the gDNA in the cells being mixed into the cell-containing solution.

In the above-mentioned mixed solution X, the content of the cell membrane impermeable compound is preferably 1 μmol/L or more, and preferably 4 μmol/L or less. When the content of the cell membrane impermeable compound is above the above-mentioned lower limit and below the above-mentioned upper limit, cell aggregation can be more effectively suppressed and the protective effect of the cell membrane can be further enhanced. Therefore, it is possible to more effectively prevent the cells from being destroyed or dying during storage, which could result in the gDNA in the cells being mixed into the cell-containing solution.

When the preservation solution contains a compound with chelating activity such as EDTA, the content of the compound with chelating activity or EDTA in the mixed solution X is preferably 4 mmol/L or more, more preferably 4.5 mmol/L or more, and preferably 7 mmol/L or less, more preferably 6.5 mmol/L or less. When the content of the compound with chelating activity or the EDTA is above the above lower limit and below the above upper limit, degradation of cfDNA can be more effectively suppressed. Furthermore, when the cell-containing solution is blood, when the content of the EDTA is above the above lower limit and below the above upper limit, coagulation of the blood during storage can be more effectively suppressed.

When the preservative solution contains a buffer such as glycine, the content of the buffer or glycine in the mixed solution X is preferably 0.1 w/v% or more, more preferably 0.2 w/v% or more, and preferably 0.5 w/v% or less, more preferably 0.4 w/v% or less. When the content of the buffer or glycine is above the lower limit and below the upper limit, the buffering effect can be effectively exerted.

In the storage container of the present invention, if the storage solution contains a formaldehyde donor compound, it is preferable that the formaldehyde content in the following mixed solution Y satisfies a specific range.

A solution Y' is obtained by mixing 7.4 g of sodium hypochlorite with 1 L of physiological saline, and an amount of the solution Y' equal to the predetermined amount of the cell-containing solution to be stored in the storage container is collected in the storage container, and the solution Y' and the storage solution are mixed to obtain a mixed solution Y. In the storage container according to the present invention, the predetermined amount of solution Y' is mixed with the storage solution contained in the storage container to obtain the mixed solution Y. Note that the solution Y' is a solution simulating a cell-containing solution.

Specifically, the above-mentioned mixed solution Y is prepared as follows.

An amount of solution Y' equal to the predetermined amount of cell-containing fluid collected in the storage container is collected in the storage container. For example, if a storage container is used to collect 5 mL of blood, 5 mL of solution Y' is collected in the storage container. The collected predetermined amount of solution Y' and the storage fluid are mixed by inversion to obtain mixed solution Y.

When the preservation solution contains a formaldehyde donor compound, in the storage container of the present invention, the formaldehyde content in the mixed solution Y is preferably 100 mg/L or more, more preferably 110 mg/L or more. When the preservation solution contains a formaldehyde donor compound, in the storage container of the present invention, the formaldehyde content in the mixed solution Y is preferably 400 mg/L or less, more preferably 300 mg/L or less, even more preferably 200 mg/L or less, and particularly preferably 190 mg/L or less. The mixed solution Y contains formaldehyde due to the formaldehyde being liberated from the formaldehyde donor compound. When the formaldehyde content in the mixed solution Y is equal to or greater than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, the formaldehyde concentration in the mixed solution of the cell-containing solution and the preservation solution can be appropriately controlled. This improves cell stability and suppresses cfDNA degradation. Furthermore, if the formaldehyde content in the above mixed solution Y exceeds 400 mg/L, cfDNAs or cfDNAs and membrane proteins are more likely to be cross-linked than when the formaldehyde content is 400 mg/L or less, which may reduce the storage stability of the cfDNA.

The formaldehyde content in the above mixed solution Y is determined by the MBTH colorimetric method.

In the storage container of the present invention, a volume of saline equal to the predetermined volume of cell-containing fluid to be stored in the storage container is collected in the storage container, and the saline and the storage fluid are mixed to obtain a mixed fluid Z. The osmotic pressure of the mixed fluid Z is preferably 300 mOsm/L or more, more preferably 350 mOsm/L or more, preferably 1100 mOsm/L or less, and more preferably 1000 mOsm/L or less. When the osmotic pressure of the mixed fluid Z is above the above lower limit and below the above upper limit, the osmotic pressure in the mixed fluid of the cell-containing fluid and the storage fluid can be improved. This allows the effects of the present invention to be exerted even more effectively.

Specifically, the above-mentioned mixed solution Z is prepared as follows.

An amount of the saline solution equivalent to the predetermined amount of cell-containing fluid to be collected in the storage container is collected in the storage container. For example, if a storage container is used to collect 5 mL of blood, 5 mL of saline solution is collected in the storage container. The predetermined amount of collected saline solution and the storage fluid are mixed by inversion to obtain mixed solution Z.

The osmotic pressure of the above mixed solution Z can be measured using an osmometer (e.g., Advanced Instruments' Osmometer 3250).

(Container body)
The shape of the container body is not particularly limited, but is preferably a bottomed container, more preferably a bottomed tubular container.

The material of the container body is not particularly limited. Examples of materials for the container body include thermoplastic resins such as polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polymethyl methacrylate, and polyacrylonitrile; thermosetting resins such as unsaturated polyester resin, epoxy resin, and epoxy-acrylate resin; modified natural resins such as cellulose acetate, cellulose propionate, ethyl cellulose, and ethyl chitin; and glass such as silicate glass, soda-lime glass, phosphosilicate glass, and borosilicate glass, and quartz glass. Only one type of material may be used for the container body, or two or more types may be used in combination.

(stopper body)
The storage container preferably includes a stopper. A conventionally known stopper can be used as the stopper. The stopper is preferably made of a material and has a shape that allows it to be attached to the opening of the container body in an airtight and liquid-tight manner. When the cell-containing fluid is blood, the stopper is preferably configured so that it can be pierced with a blood collection needle.

Examples of the above-mentioned stopper include a stopper having a shape that fits into the opening of the container body, a sheet-shaped sealing stopper, etc.

The stopper may also be a stopper comprising a stopper body such as a rubber stopper and a cap member made of plastic or the like. In this case, the risk of blood coming into contact with the human body can be reduced when the stopper body is pulled out from the opening of the container body after blood collection.

Examples of the material for the stopper (or the stopper main body) include synthetic resin, elastomer, rubber, metal foil, etc. Examples of the rubber include butyl rubber and halogenated butyl rubber. Examples of the metal foil include aluminum foil. From the perspective of improving sealing performance, the material for the stopper is preferably butyl rubber. The stopper is preferably a butyl rubber stopper.

(Other details of storage container and storage solution)
The storage container is a container for storing a predetermined amount of cell-containing fluid. The storage container is a container for collecting and storing a predetermined amount of cell-containing fluid. The storage container is a container for storing the cell-containing fluid and the preservation fluid in a mixed state. The predetermined amount of the cell-containing fluid is changed as appropriate depending on the size of the storage container, etc. The predetermined amount of the cell-containing fluid is, for example, 1 mL, 5 mL, 10 mL, or 20 mL.

The storage container is preferably a container for storing the cell-containing liquid in liquid form, and is preferably a container for storing the cell-containing liquid without freezing. The storage container is preferably a container for storing the cell-containing liquid at 0°C or higher, more preferably a container for storing the cell-containing liquid at 1°C or higher, even more preferably a container for storing the cell-containing liquid at 15°C or higher, and particularly preferably a container for storing the cell-containing liquid at 18°C or higher. The storage container is preferably a container for storing the cell-containing liquid at 100°C or lower, more preferably a container for storing the cell-containing liquid at 50°C or lower, even more preferably a container for storing the cell-containing liquid at 25°C or lower, and particularly preferably a container for storing the cell-containing liquid at 24°C or lower.

The preservation solution is preferably used to store the cell-containing liquid at 0°C or higher, more preferably at 1°C or higher, even more preferably at 15°C or higher, and particularly preferably at 18°C or higher. The preservation solution is preferably used to store the cell-containing liquid at 100°C or lower, more preferably at 50°C or lower, even more preferably at 25°C or lower, and particularly preferably at 24°C or lower.

For each 1 mL of cell-containing fluid to be stored, the amount of preservation fluid contained in the container body is preferably 0.05 mL or more, more preferably 0.1 mL or more, and preferably 1 mL or less, more preferably 0.5 mL or less. When the amount of preservation fluid is above the lower limit and below the upper limit, the cell-containing fluid is not excessively diluted, and the effects of the present invention can be more effectively achieved.

The above-mentioned preservation solution is preferably used by mixing 0.05 mL or more, more preferably 0.1 mL or more, and preferably 1 mL or less, and more preferably 0.5 mL or less, per 1 mL of cell-containing solution to be preserved. In this case, the cell-containing solution is not excessively diluted, and the effects of the present invention can be more effectively achieved.

The internal pressure of the storage container is not particularly limited. The storage container can also be used as a vacuum blood collection tube, sealed with the sealing member after the interior has been evacuated. When used as a vacuum blood collection tube, a fixed amount of blood can be easily collected regardless of the skill level of the blood collector.

In order to prevent bacterial infection, it is preferable that the inside of the storage container be sterilized in accordance with ISO and JIS standards.

The present invention will be described in more detail below with reference to examples. The present invention is not limited to the following examples. Examples 3 to 15 are for reference only.

The following ingredients were prepared for the preservative solution.

(Cell membrane permeable compound)
Propylene glycol Glycerin Dimethyl sulfoxide

(Compound that does not permeate cell membranes)
Polyethylene glycol (weight average molecular weight 500,000)
Polyvinylpyrrolidone (weight average molecular weight 30,000)
Dextran (weight average molecular weight 200,000)

(Formaldehyde donor compounds)
1-hydroxymethyl-5,5-dimethylhydantoin

(osmotic pressure adjusting agent)
glucose

(anticoagulant)
EDTA 2Na

(Buffering agent)
glycine

(others)
water

(Examples 1 to 15)
Preparation of storage solution:
The ingredients shown in Tables 1 to 4 were mixed in the amounts shown in Tables 1 to 4 to obtain preservative solutions.

Preparation of storage container:
A polyethylene terephthalate (PET) bottomed tube with a length of 100 mm and an inner diameter of 14 mm at the opening was prepared as the container body. 0.5 mL of the obtained preservation solution was placed in the PET bottomed tube. The pressure inside the preservation container was reduced to 50 kPa, and the container was sealed with a butyl rubber stopper. In this way, a preservation container for storing 5 mL of blood was prepared.

(Comparative Examples 1 and 4)
A vacuum blood collection tube containing EDTA ("Insepack II-D" manufactured by Sekisui Medical Co., Ltd.) was used as a storage container. This vacuum blood collection tube did not contain a cell membrane-permeable compound, a cell membrane-impermeable compound, or a formaldehyde donor compound.

(Comparative Examples 2 and 3)
A preservative solution and a preservative container were obtained in the same manner as in Example 1, except that the types and contents of the blended components were changed as shown in Table 1.

(evaluation)
(1) Contents of cell membrane-permeable compound, cell membrane impermeable compound, formaldehyde donor compound, osmotic pressure adjuster, anticoagulant, and buffer in mixed solution X 5 mL of blood was added to the storage containers obtained in Examples 1 to 15 and Comparative Examples 2 and 3, and the blood and storage solution were mixed by inversion to obtain mixed solution X. The contents of the cell membrane-permeable compound, cell membrane impermeable compound, formaldehyde donor compound, osmotic pressure adjuster, anticoagulant, and buffer in the obtained mixed solution X were determined.

(2) Formaldehyde Content in Mixed Solution Y A solution Y' was prepared by mixing 7.4 g of sodium hypochlorite with 1 L of physiological saline. 5 mL of solution Y' was added to the storage containers obtained in Examples 1 and 2 and Comparative Examples 2 and 3, and solution Y' and the storage solution were mixed by inversion to obtain mixed solution Y. The formaldehyde content in the obtained mixed solution Y was determined by MBTH colorimetry.

(3) Osmotic Pressure of Mixture Z 5 mL of physiological saline was added to the storage containers obtained in Examples 1 to 15 and Comparative Example 2, and the physiological saline and storage solution were mixed by inversion to obtain mixture Z. The osmotic pressure of the obtained mixture Z was measured using an osmometer (Advanced Instruments'"Osmometer3250").

(4) Storage stability of cfDNA (4-1) Storage stability of cfDNA stored at room temperature for 7 days (Examples 1 and 2 and Comparative Examples 1 to 3)
Five mL of blood was collected into the storage containers obtained in Examples 1 and 2 and Comparative Examples 1 to 3, and the blood and storage solution were mixed by inversion. The storage containers were then left to stand in an environment of 15°C to 25°C. Immediately after storage, after 3 days, and after 7 days, the mixture of blood and storage solution was collected from the storage container. Plasma was recovered from the collected mixture by centrifugation. DNA contained in the collected plasma was purified using a cfDNA purification kit (QIAGEN "QIAamp Circulating Nucleic Acid Kit").

The purified DNA was fluorescently labeled and then electrophoresed using an electrophoresis system (Agilent 2100 Bioanalyzer and High Sensitivity DNA Kit, manufactured by Agilent). The electrophoresis images were analyzed to observe the peak shapes of cfDNA around 180 bp and gDNA above 300 bp.

(4-2) Storage stability of cfDNA at room temperature for 1 day (Examples 3 to 12 and Comparative Example 4)
The test was conducted in the same manner as in "(4-1) Storage stability of cfDNA stored at room temperature for 7 days," except that the storage containers obtained in Examples 3 to 12 and Comparative Example 4 were used, and the mixed solution of blood and storage solution was collected after the storage containers were left to stand in an environment of 15°C to 25°C for 1 day.

(4-3) Storage stability of cfDNA at room temperature for 4 days (Examples 13 to 15)
The test was conducted in the same manner as in "(4-1) Storage stability of cfDNA stored at room temperature for 7 days," except that the storage containers obtained in Examples 13 to 15 were used and the mixture of blood and storage solution was collected after leaving the storage containers undisturbed for 3 days and after leaving them undisturbed for 4 days in an environment of 15°C to 25°C.

The compositions and results are shown in Tables 1 to 4 and Figures 1 to 11 below.

In Figures 1 to 11, the horizontal axis represents the number of DNA base pairs (bp), and the vertical axis represents fluorescence intensity. In Figures 1 to 11, the peaks indicated by M1 and M2 are marker peaks, and the peak indicated by S is a peak derived from cfDNA.

Figures 1(a) to 1(c) show the results of image analysis of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Example 1. Figures 2(a) and 2(b) show the results of image analysis of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Example 2. Figures 3(a) to 3(c) show the results of image analysis of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Comparative Example 1. Figures 4(a) and 4(b) show the results of image analysis of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Comparative Example 2. Figures 5(a) to 5(c) show the results of image analysis of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Comparative Example 3. Figures 1(a), 2(a), 3(a), 4(a), and 5(a) show the results obtained using the mixed solution immediately after blood was collected in the storage container. Figures 1(b), 3(b), 4(b), and 5(b) show the results obtained using the mixed solution three days after blood had been stored in the storage container. 1(c), 2(b), 3(c), and 5(c) show the results obtained using the mixed solution after 7 days of storing the blood in the storage container.

As shown in Figures 1 and 2, for the purified DNA obtained in Examples 1 and 2, no peaks other than the cfDNA-derived peak around 180 bp were observed up to the seventh day of storage. Furthermore, the shape of the cfDNA-derived peak was well maintained over the seven days after storage in an environment between 15°C and 25°C, demonstrating the enhanced storage stability of cfDNA.

On the other hand, as shown in Figures 3 and 4, peaks of 300 bp or more derived from DNA other than cfDNA were detected in the purified DNA obtained in Comparative Examples 1 and 2. These peaks are thought to be due to gDNA contaminating the blood from white blood cells, etc.

Furthermore, as shown in Figure 5, although the contamination of gDNA from white blood cells and other sources was suppressed to some extent in the purified DNA obtained in Comparative Example 3, it can be seen that the cfDNA peak broadened and the fluorescence intensity decreased. This is presumably due to cross-linking of cfDNA molecules by formaldehyde.

Figures 6(a) to (c) show the image analysis results of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Examples 3 to 5, respectively. Figures 7(a) to (c) show the image analysis results of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Examples 6 to 8, respectively. Figures 8(a) to (d) show the image analysis results of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Examples 9 to 12, respectively. Figure 3(d) shows the image analysis results of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Comparative Example 4. Figures 6(a) to (c), Figures 7(a) to (c), Figures 8(a) to (d), and Figure 3(d) show the results obtained using the mixed solution one day after blood had been stored in the storage container.

As shown in Figure 6, in Examples 3 to 5, peaks were detected due to gDNA contamination of the blood from white blood cells, etc., but the storage stability of cfDNA was improved compared to Comparison Example 4.

As shown in Figure 7, the storage stability of cfDNA was improved in Examples 6 to 8.

As shown in Figures 8(a) and (b), the storage stability of cfDNA was improved in Examples 9 and 10 compared to Comparison Example 4.

As shown in Figures 8(c) and (d), the storage stability of cfDNA was improved in Examples 11 and 12.

Figures 9(a) and (b) show the results of image analysis of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Example 13. Figures 10(a) and (b) show the results of image analysis of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Example 14. Figures 11(a) and (b) show the results of image analysis of electrophoresis photographs obtained in the evaluation of the storage stability of cfDNA in Example 15. Figures 9(a), 10(a), and 11(a) show the results obtained using the mixed solution three days after storing blood in a storage container. Figures 9(b), 10(b), and 11(b) show the results obtained using the mixed solution four days after storing blood in a storage container.

M1, M2...markers S...cfDNA

Claims (14)

A container for storing a predetermined amount of cell-containing liquid,
A container body and a preservative solution contained in the container body,
a first configuration in which the preservation solution contains a cell membrane-permeable compound having a molecular weight of 100 or less and not freezing at 0°C, and when the predetermined amount of cell-containing solution is collected in a cell-containing solution storage container and the cell-containing solution and the preservation solution are mixed to obtain a mixed solution X, the content of the cell membrane-permeable compound in the mixed solution X is 1 vol% or more and 5 vol% or less ;
a second configuration in which the preservation solution contains a cell membrane impermeable compound having a molecular weight of 300 or more, and when the predetermined amount of cell-containing solution is collected in a cell-containing solution storage container and the cell-containing solution and the preservation solution are mixed to obtain a mixed solution X, the content of the cell membrane impermeable compound in the mixed solution X is 0.5 μmol/L or more and 5 μmol/L or less ;
the cell membrane-permeable compound is ethylene glycol, propylene glycol, glycerin, dimethyl sulfoxide, acetamide, 1,3-propanediol, or butylene glycol;
the cell membrane impermeable compound is polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, polysaccharides, hydroxypropyl cellulose, sugar alcohol, or ficoll;
A storage container for a cell-containing liquid , wherein the storage liquid contains a formaldehyde donor compound, and the formaldehyde donor compound is DMDM hydantoin or 1-hydroxymethyl-5,5-dimethylhydantoin . 2. The storage container for cell-containing liquid according to claim 1 , wherein the cell membrane impermeable compound is a compound having a molecular weight of 1,000 or more. 3. The storage container for cell-containing fluid according to claim 1 or 2, wherein when a solution Y' is obtained by mixing 7.4 g of sodium hypochlorite with 1 L of physiological saline, and an amount of the solution Y' equal to a predetermined amount of the cell-containing fluid to be stored in the storage container for cell-containing fluid is collected in the storage container for cell-containing fluid, and a mixed solution Y is obtained by mixing the solution Y' and the storage fluid, the formaldehyde content in the mixed solution Y is 100 mg/L or more and 400 mg/L or less. 4. The cell-containing fluid storage container according to claim 1, wherein when a volume of physiological saline equal to a predetermined volume of the cell-containing fluid to be stored in the cell-containing fluid storage container is collected in the cell-containing fluid storage container and the physiological saline and the storage fluid are mixed to obtain a mixed fluid Z, the osmotic pressure of the mixed fluid Z is 300 mOsm/ L or more and 1100 mOsm/L or less. the preservative solution comprises an osmolality adjusting agent;
5. The storage container for a cell-containing liquid according to claim 1 , wherein the osmotic pressure adjusting agent is glucose or sodium chloride. the preservative solution contains a compound having a chelating effect,
the chelating compound comprises EDTA;
6. The cell-containing liquid storage container according to claim 1 , wherein the content of the EDTA in the mixed liquid X is 4 mmol/L or more and 7 mmol/L or less. the storage solution comprises a buffer ;
the buffer comprises glycine;
7. The storage container for cell-containing liquid according to claim 1 , wherein the content of the glycine in the mixed liquid X is 0.5 w/v % or less. The storage container for a cell-containing liquid according to any one of claims 1 to 7 , wherein the cell-containing liquid is blood. A preservation solution used to preserve a cell-containing solution,
A third configuration, which includes a cell membrane-permeable compound having a molecular weight of 100 or less and not freezing at 0°C, and the content of the cell membrane-permeable compound is 2 vol% or more and 50 vol% or less;
a fourth configuration in which the cell membrane impermeable compound has a molecular weight of 300 or more, and the content of the cell membrane impermeable compound is 1.0 μmol/L or more and 100 μmol/L or less ;
the cell membrane-permeable compound is ethylene glycol, propylene glycol, glycerin, dimethyl sulfoxide, acetamide, 1,3-propanediol, or butylene glycol;
the cell membrane impermeable compound is polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, polysaccharides, hydroxypropyl cellulose, sugar alcohol, or ficoll;
The preservation solution comprises a formaldehyde donor compound, and the formaldehyde donor compound is DMDM hydantoin or 1-hydroxymethyl-5,5-dimethylhydantoin . The preservation solution according to claim 9 , wherein the cell membrane-impermeable compound is a compound having a molecular weight of 1,000 or more. Contains an osmolality adjuster,
The preservative solution according to claim 9 or 10 , wherein the osmotic agent is glucose or sodium chloride. Contains a compound with chelating activity,
The preservative solution according to any one of claims 9 to 11 , wherein the compound having chelating activity comprises EDTA. Contains a buffer ,
The preservation solution according to any one of claims 9 to 12 , wherein the buffer comprises glycine. The preservation solution according to any one of claims 9 to 13 , wherein the cell-containing solution is blood.